Real-time PCR assay for detecting Lactobacillus plantarum group using species/subspecies-specific genes identified by comparative genomics

Abstract

The Lactobacillus plantarum group is composed of four closely related species or subspecies. To clarify the L. plantarum group classification and accurately provide health benefits of probiotics, it is important to distinguish them correctly. In this study, species- or subspecies-specific genes were identified to distinguish the L. plantarum group, and a real-time polymerase chain reaction (PCR) assay was developed to accurately detect them. Specific genes, such as protein-coding genes present in all strains of target species or subspecies but not in other bacterial strains, were explored from 70 genome sequences using comparative genomics. As a result, genes encoding a transporter, major facilitator family protein, and hypothetical proteins were confirmed as specific genes. Specificity of primers was evaluated with 55 lactic acid bacteria, and all primers showed 100% specificity. Probiotic products and fermented food samples were used to validate the developed real-time PCR assay. Of the probiotic products, one product was found to contain L. pentosus, even though it was labeled to contain L. plantarum. Using our in-house developed real-time PCR assay, the products labeled as L. plantarum were identified at the subspecies level, and the L. plantarum group species or subspecies in fermented food samples were successfully qualified and quantified.